Figure 2. The effects of PPT1 inhibition are not recapitulated by Ulk1, Pik3c3, and Atg7 KD or Atg7 KO, and HCQ does not impair CD8⁺ T cell–mediated killing.

(A) Schematic of priming and coculture experiments to measure antigen-specific T cell killing in vitro. (B) One hundred percent lactate dehydrogenase (LDH) from the coculture of primed or unprimed splenocytes with live B16, in the presence or absence of HCQ (10 μM). Each experiment was performed in triplicates, and results were reproduced with 3 independent experiments. Concanavalin A was used as a nonspecific splenocyte priming agent. (C) One hundred percent LDH measurement in primed splenocytes cocultured with B16 with/without indicated treatments. (D) Immunoblots confirming the B16 Ulk1-, Pik3c3-, and Atg7-KD status. (E) Dot plot representing ELISA performed for the measurement of splenocyte-secreted IFN-γ upon coculturing with B16 with Ulk1-, Pik3c3-, and Atg7-KD conditions. Each experiment was performed in triplicates, and the results were reproduced by 3 independent experiments. (F) Immunoblot confirming the B16 Ppt1-KD status. (G) Dot plot representing IFN-γ ELISA in Ppt1-KD B16 coculture as above in E for 3 independent experiments. (H) Measurement of percentage T cell–mediated cytotoxicity of B16 cells in Ulk1, Pik3c3, Atg7, and Ppt1 condition. (I) Immunoblot confirming the B16 Atg7-KO status. (J) Irradiated B16-primed splenocytes or purified splenic CD8⁺ T cells were cocultured with B16 WT Atg7 or B16 Atg7-KO cells, and the percentage proliferation was measured by performing 3 independent experiments. (K) B16 Cas9 control or B16 Atg7-KO cells (5 × 10⁵) were injected into the flanks of C57BL6/J mice. After tumors reached a size of 50 mm³, mice were randomized to cohorts of n = 5 mice each, and B16 Cas9 control tumors were treated with IgG control + vehicle, anti–PD-1 Ab (200 μg i.p. every other day) + vehicle, IgG + HCQ (60 mg/kg i.p. daily), or the combination. B16 Atg7-KO tumors were treated with either IgG control or anti–PD-1 Ab. The average growth rate ± SEM is shown. (L) Tumors and spleens were harvested from the experiment in B, on day 8 of treatment. The percentage of CD8⁺ T cells in CD45⁺ cells in spleen and tumor are shown. All t tests were 2 tailed (G and K). One-way ANOVA and Bonferroni’s adjustment (B, C, and J) or Dunnett’s procedure (E and L). E:T, effector/target; sgNT, single guide nontargeting.